Chemical approaches for synthesis and engineering of proteins offer many advantages to recombinant methods in that one can incorporate non-natural or selectively labelled amino acids. However, peptide synthesis is practically limited to small proteins (typically &lt;50 residues) due to the accumulation of side-products and racemization that complicate product purification and decrease yields (for recent reviews see Kaiser, E. T. (1989) Acc. Chem. Res. 22, 47-54; Offord, R. E. (1987) Prot. Eng. 1, 151-157).
Proteolytic enzymes, in particular serine proteases, have reportedly been used as alternatives to synthetic peptide chemistry because of their stereoselective properties and mild reaction conditions (for reviews see Kullman, W. (1987) In: Enzymatic Peptide Synthesis, CRC Press, Florida U.S.; Chaiken, (1981) CRC Crit. Rev. Biochem. 11, 255-301). Such enzymes reportedly have been used to complement chemical coupling methods to produce larger peptides by blockwise enzymatic coupling of synthetic fragments. Inouye et al. (1979), J. Am. Chem. Soc., 101, 751-752 (insulin fragments); Hommandberg and Laskowski, (1979) Biochemistry 18, 586-592 (ribonuclease fragments)). However, the narrow substrate specificities and intrinsic hydrolytic (peptidase) activity of serine proteases have limited their use in peptide synthesis.
A central problem in the case of serine proteases in peptide synthesis is that hydrolysis of the acyl-enzyme intermediate is strongly favored over aminolysis (FIG. 1). Several laboratories have reported that the equilibrium is shifted from hydrolysis toward aminolysis by use of mixed or pure organic solvents to carry out catalysis (Coletti-Previero et al., (1969) J. Mol. Biol. 39, 493-501; Barbas et al., (1988) J. Am. Chem. Soc. 110, 5162-5166). However, enzymes are generally less stable and relatively insoluble in organic solvents (Wong et al., (1990) J. Am. Chem. Soc. 112, 945-953; Klibanov, (1986) Chemtech 16, 354-359). Further, kinetic activation barriers in organic solvents are higher for the charged transition-states involved leading to lower enzymatic activity. In an attempt to avoid these problems, one laboratory reported that thiolsubtilisin, a derivative of the bacterial serine protease in which the active site Ser221 was chemically converted to a Cys (S221C), shifted the preference for aminolysis to hydrolysis by &gt;1000-fold for very small peptides. Nakasuta et al. (1987) J. Am. Chem. Soc. 109, 3808-3810.
This shift was attributed to the kinetic preference of thioesters to react with amines over water. Based upon similar principles, another laboratory reported that selenolsubtilisin had a 14,000-fold shift in preference for aminolysis over hydrolysis. Wu and Hilvert (1989) J. Am. Chem. Soc. 111, 4513-4514. However the catalytic efficiencies for aminolysis of a chemically activated ester by either thiol- or selenolsubtilisin are about 10.sup.3 - and 10.sup.4 -fold, respectively, below the esterase activity of wild-type subtilisin. Although chemically active esters have reportedly been used to increase the rates for acylation of thiol- or selenolsubtilisin (e.g. the acylation of thiolsubtilisin with a p-chlorophenyl ester of an 8-mer peptide for ligation with a 4-mer peptide in &gt;50% DMF), such activated esters present synthetic difficulties as well as creating substrates prone to spontaneous hydrolysis in aqueous solvents (Nakatsuka et al. (1987) supra.).
The serine proteases comprise a diverse class of enzymes having a wide range of specificities and biological functions. Stroud, R. M. (1974) Sci Amer. 131, 74-88. Despite their functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct families of enzymes: the Bacillus subtilisin-type serine proteases and the mammalian and homologous bacterial trypsin-type serine proteases (e.g., trypsin and S. gresius trypsin). These two families of serine proteases show remarkably similar mechanisms of catalysis. Kraut, J. (1977) Ann. Rev. Biochem., 46, 331-358. Furthermore, although the primary structure is unrelated, the tertiary structure of these two enzyme families bring together a conserved catalytic triad of amino acids consisting of serine, histidine and aspartate.
Subtilisin is a serine endoprotease (MW.sup.- 27,500) which is secreted in large amounts from a wide variety of Bacillus species. The protein sequence of subtilisin has been determined from at least four different species of Bacillus. Markland, F. S., et al. (1971) in The Enzymes, ed. Boyer, P. D., Acad. Press, New York, Vol. III, pp. 561-608; Nedkov, P. et al. (1983) Hoppe-Seyler's Z. Physiol. Chem., 364, 1537-1540. The three-dimensional crystallographic structure of subtilisin BPN' (from B. amyloliquefaciens) to 2.5.ANG. resolution has also been reported. Bott, et al. (1988), J. Biol. Chem., 263, 7895-7906; McPhalen, et al. (1988), Biochemistry, 27, 6582-6598; Wright, C. S., et al. (1969), Nature, 221,235-242; Drenth, J. et al. (1972) Eur. J. Biochem.,26,177-181. These studies indicate that although subtilisin is genetically unrelated to the mammalian serine proteases, it has a similar active site structure. The x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, J. D., et al. (1972), Biochemistry, 11, 2439-2449), product complexes (Robertus, J. D., et al. (1972) Biochemistry 11, 4293-4303), and transition state analogs (Matthews, D. A., et al (1975) J. Biol. Chem. 250, 7120-7126; Poulos, T. L., et al. (1976) J. Biol. Chem. 251, 1097-1103), which have been reported have also provided information regarding the active site and putative substrate binding cleft of subtilisin. In addition, a large number of kinetic and chemical modification studies have been reported for subtilisin (Philipp, M., et al. (1983) Mol. Cell. Biochem. 51, 5-32; Svendsen, I. B. (1976) Carlsberg Res. Comm. 41, 237-291; Markland, F. S. Id.). Stauffer, D. C., et al. (1965) J. Biol. Chem. 244, 5333-5338; Polgar, L. et al. (1981) Biochem. Biophys. Acta 667,351-354).
U.S. Pat. No. 4,760,025 discloses subtilisin mutants wherein a different amino acid is substituted for the naturally-occurring amino acid residues of Bacillus amyloliquifaciens subtilisin at positions +32, +155, +104, +222, +166, +64, +33, +169, +189, +217, or +156.
The references discussed above are provided solely for their disclosure prior to the filing date of the present case, and nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or priority based on earlier filed applications.